Methods of use of sialoadhesin factor-2 antibodies

ABSTRACT

Monoclonal antibodies have been generated that bind to human sialoadhesion factor-2. These antibodies are useful as diagnostic and therapeutic reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/546,586, filed Oct. 12, 2006, now U.S. Pat. No. 7,871,612, which is aDivisional of U.S. application Ser. No. 10/232,187, filed Aug. 29, 2002,now abandoned, which is a Continuation-in-part of InternationalApplication No PCT/US2001/007193, filed Mar. 5, 2001, now abandoned,which claims the benefit of U.S. Provisional Application No. 60/187,595,filed Mar. 7, 2000. The entire contents of the aforementioned patentapplications are hereby incorporated herein by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention resulted from research funded in whole or in part by theNational Institutes of Health, Grant No. AI41472. The Federal Governmenthas certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 19, 2009 andmodified on May 17, 2011, is named PCTSEQLIST.TXT and is 4,324 bytes insize.

FIELD OF THE INVENTION

This invention relates to monoclonal antibodies (mAbs) that bind tosialoadhesin factor-2 (SAF-2) and to the use of such antibodies fordiagnostic and therapeutic purposes. This invention also relates to theprevention and treatment of diseases and conditions mediated by cellsexpressing SAF-2.

BACKGROUND OF THE INVENTION

Eosinophils, basophils and mast cells have been implicated as the majorcell types producing inflammatory mediators in response to helminthicinfections, as well as several diseases, particularly asthma, rhinitis,and atopic dermatitis (Weller, P. F. (1991) N. Engl. J. Med. 324:1110;Sur, S., C. et al. (1993) In Allergy Principles and Practice. E.Middleton et al. eds. Mosby, St. Louis, Mo., p. 169; Costa, J. J. et al.(1997) JAMA 278:1815). In these situations, the preferentialaccumulation and activation of these cells has been noted. Althoughconsiderable progress has been made in our understanding of eosinophilrecruitment to the site of inflammation, a number of key points arestill unclear, including the exact mediators utilized for localizationto these sites during the migration process. For example, activation ofmicrovascular endothelial cells and expression of adhesion molecules,notably VCAM-1, is felt to be a key event in this process duringallergic inflammation (Bochner, B. S. (1998) In Allergy Principles andPractice. J. Middleton et al. eds. Mosby, St. Louis). In addition, anumber of chemokines and other chemotactic factors, such as those actingvia CCR3, have been implicated because of their involvement ineosinophil, basophil and mast cell chemotaxis (Dahinden, C. A. et al.(1994) J. Exp. Med. 179:751; Daffern, P. J. et al. (1995) J. Exp. Med.181:2119; Nickel, R., L. et al. (1999) J. Allergy Clin. Immunol.104:723; Romagnani, P. et al. (1999) Am. J. Pathol. 155:1195; Rot, A. etal. (1992) J. Exp. Med. 176:1489). Another possibility, however, is thatthese cells are selectively recruited and activated in a specific waydue to a unique cell surface phenotype. While eosinophils, basophils andmast cells are readily identifiable based on their tinctorialproperties, as yet there has been no cell surface marker identified thatis unique to these cell subsets (Saito, H. et al. (1986) Blood 67:50;Bodger, M. P. et al. (1987) Blood 69:1414).

Sialoadhesin factor-2, or SAF-2 (European Patent Publication No. EP 0924 297 A1), is a member of the sialoadhesin family of proteins alsoknown as the I-type lectins and recently renamed the siglec family(sialic acid-binding Ig-like lectins) (Kelm, S. et al. (1996)Glycoconjugate Journal 13:913). The family members include sialoadhesin(siglec-1), CD22 (siglec-2), CD33 (siglec-3), myelin associatedglycoprotein (MAG or siglec-4), siglec-5 (Cornish, A. L. et al. (1998)Blood 92:2123), OB-BP-1/siglec-6 (Patel, N. et al. (1999) J. Biol. Chem.274:22729) and AIRM1 or siglec-7 (Falco, M. et al. (1999)J. Exp. Med.190:793; Nicoll, G. et al. (1999) J. Biol. Chem. 274:34089). With theexception of siglec-4, all are expressed on various subsets ofhematopoietic cells. Siglecs belong to the immunoglobulin (Ig) supergenefamily and have an N-terminal V-set Ig domain followed by 1-16 C2 set Igdomains. Siglecs mediate sialic acid-dependent adhesion with other cellsgenerally preferring either α2,3 linkages (siglec-1, -3, and -4) or α2,6linkages (siglec-2) (Kelm et al. supra). Most family members have eitherimmunoreceptor tyrosine-based inhibition motifs (ITIM) or activationmotifs (ITAM) that participate in signaling through Src homology 2 (SH2)domain binding to the phosphotyrosine of the ITIM or ITAM. This has beendemonstrated for CD22, CD33 and AIRM1 (Falco et al., supra; Freeman, S.D. et al. (1995) Blood 85:2005; Blasioli, J. et al. (1999) J. Biol.Chem. 274:2302).

SAF-2, now known as Siglec-8, exists in two isoforms with identicalextracellular and transmembrane sequences. One isoform has a shortcytoplasmic tail with no known signaling sequences (Siglec-8), while theother, Siglec-8 long form (Siglec-8L), has a longer cytoplasmic tailcontaining two tyrosine-based signaling motifs (Foussias, G. et al.(2000) Biochem Biophys Res Commun 278:775; Munday, J. et al. (2001)Biochem. J. 355:489). Although the function of Siglec-8 and Siglec-8L,and indeed most Siglecs, is unknown, the cytoplasmic region of Siglec-8Lcontains one consensus immunoreceptor tyrosine-based inhibitory motif(ITIM) and a signaling lymphocyte activation molecule (SLAM)-like motif,suggesting that Siglec-8L may possess signal transduction activity.

SUMMARY OF THE INVENTION

One aspect of the present invention includes an antibody that binds tohuman SAF-2. More specifically, the present invention includes amonoclonal antibody having the identifying characteristics of monoclonalantibody 2C4. A specific embodiment of this aspect of the presentinvention is an antibody comprising a heavy chain variable regionpolypeptide as set forth in SEQ ID NO:2 and a kappa light chain variableregion polypeptide as set forth in SEQ ID NO:4.

The present invention also includes an immunoglobulin heavy chaincomplementarity determining region comprising any of the polypeptidesset forth in SEQ ID NOs:5, 6 or 7 or any combination thereof, and animmunoglobulin kappa light chain complementarity determining regioncomprising any of the polypeptides set forth in SEQ ID NOs:8, 9 or 10 orany combination thereof. A preferred embodiment of the present inventionis a polypeptide comprising an immunoglobulin complementaritydetermining region comprising the polypeptides set forth in SEQ IDNOs:5, 6, 7, 8, 9 and 10. The present invention also includes anisolated polynucleotide encoding any of the forgoing polypeptides.

An additional embodiment of the present invention is a method fordetecting the presence of a cell in a sample wherein the cell comprisesan SAF-2 protein, the method comprising a) exposing the sample to anantibody that binds to SAF-2 and b) detecting the antibody that is boundto SAF-2. The sample suspected of containing the cell can optionally betreated before exposure to the antibody in order to render the SAF-2susceptible to binding by the antibody. The preferred utility for thisembodiment is the detection of eosinophils.

Another aspect of the instant invention is a method for the preventionand treatment of a disease and condition mediated by cells expressingSAF-2, the method comprising administering to a subject in need thereofan effective amount of a pharmaceutical composition that comprises atherapeutic agent that binds to SAF-2. Preferred is a method fortreating or preventing an allergic, asthmatic or cancerous diseasestate, as well as hypereosinophilic syndromes. Most preferred is amethod for preventing or treating asthma, allergic rhinitis, nasalpolyposis, atopic dermatitis, chronic urticaria, mastocytosis oreosinophilic or basophilic leukemias. A preferred therapeutic agent thatcomprises the composition for use in the method is a monoclonal antibodyor fragment thereof which binds to human SAF-2 and has the identifyingcharacteristics of monoclonal antibody 2C4.

Yet another aspect of the present invention includes a pharmaceuticalcomposition comprising an effective amount of a therapeutic agent thatbinds to SAF-2. A preferred therapeutic agent is a monoclonal antibodyagainst human SAF-2 having the identifying characteristics of monoclonalantibody 2C4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the V_(H) cDNA sequence and the deduced amino acid sequenceof a monoclonal antibody that binds to SAF-2, mAb 2C4 (SEQ ID NOs:1 and2, respectively). The bolded residues indicate the three CDRs (SEQ IDNOs:5, 6, and 7).

FIG. 2 shows the V_(K) cDNA sequence and the deduced amino acid sequenceof a monoclonal antibody that binds to SAF-2, 2C4 (SEQ ID NOs:3 and 4,respectively). The bolded residues indicate the three CDRs (SEQ IDNOs:8, 9, and 10).

FIG. 3 presents data on expression of SAF-2 on human peripheral bloodeosinophils, basophils and 16 week old cord blood-derived cultured mastcells. Histograms shown are representative of 3-4 experiments withvirtually identical results for each cell type. Monoclonal reagents usedas positive and negative controls are also shown.

FIG. 4 shows the effect of Siglec-8 crosslinking on eosinophil death.Purified peripheral blood eosinophils were cultured under the indicatedconditions. Viability was assessed using erythrosin-B dye exclusion.Data are from six experiments.

FIG. 5 demonstrates that Siglec-8 ligation induces eosinophil apoptosis.Eosinophils were cultured as indicated, harvested and analyzed by flowcytometry for annexin-V labeling. Data are from six experiments.

FIG. 6 demonstrates the effect of IL-5 and GM-CSF on Siglec-8crosslinking-induced eosinophil death. In panel a, IL-5 (1 ng/ml) wasadded simultaneously at the beginning of the cell culture and viabilitydetermined at various time points as indicated. Data are from 4-6experiments. In panel b, IL-5 or GM-CSF (each used at 30 ng/ml) reducesthe concentration of Siglec-8 mAb needed to induce maximal eosinophilapoptosis. Eosinophils were initially cultured with IL-5 or GM-CSF inthe presence of secondary Ab and the indicated concentrations of 2E2.After 24 h, apoptosis was analyzed using annexin-V staining. Data arepresented as mean±SD, n=2.

FIG. 7 demonstrates that IL-5 or GM-CSF priming enhances eosinophilapoptosis in response to Siglec-8 mAb. Eosinophils were preincubatedwith or without IL-5 or GM-CSF (each at 30 ng/ml) for 24 h. Antibodieswere then added to the cultures, as indicated, and apoptosis wasanalyzed using annexin-V staining 24 h later. Data are presented asmean±SD of two experiments.

FIG. 8 demonstrates that monoclonal antibodies to Siglec-8 either aloneor in the presence of a secondary, crosslinking antibody (pc) inducescellular death in eosinophils obtained from bronchoalveolar lavage fluidafter segmental allergen challenge. Siglec-8 antibody (2E2 Ab) was usedat two different concentrations: 2.5 and 10 ug/ml (n=1, data presentedas mean+/−SEM of a duplicate set of experiments).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a variety of antibodies, includingaltered antibodies and fragments thereof, directed against SAF-2, whichare characterized by their ability to bind to human SAF-2 polypeptide orpolypeptides derived therefrom. Exemplary of this class of antibodies ismonoclonal antibody 2C4. These antibody products are useful in thedetection of cells comprising SAF-2 polypeptide including the specificdetection of eosinophils. These antibody products are also useful intherapeutic and pharmaceutical compositions for treating allergicrhinitis, allergies, asthma, eczema, or diseases such as lymphoma,leukemia, or systemic mastocytosis. Alternatively, the antibodies of theinvention can be coupled to toxins, antiproliferative drugs orradionuclides to kill cells in areas of excessive SAF-2 expression,thereby treating allergic rhinitis, allergies, asthma, eczema, ordiseases such as lymphoma, leukemia, or systemic mastocytosis.

The instant invention also provides a novel means to treat or preventvarious disease states that are mediated by cells (or molecules by suchcells) expressing SAF-2. These disease states include various allergies,asthma and cancers. The instant invention pertains to the findings thatSAF-2 represents a unique cell surface marker for a circumscribed set ofcells (eosinophils, basophils and mast cells), and that binding oftherapeutic agent, such as an antibody or an altered antibody (asdefined herein), results in the specific reduction in such cells thatmediate such disease states.

“Therapeutic agent” refers to a prophylactically or therapeuticallyeffective molecule, including a polypeptide, an antibody or alteredantibody, and an agonist/antagonist peptide or small molecule compound.

“Antibodies” refers to immunoglobulins which can be prepared byconventional hybridoma techniques, phage display combinatoriallibraries, immunoglobulin chain shuffling and humanization techniques.Also included are fully human monoclonal antibodies. As used herein,“antibody” also includes “altered antibody” which refers to a proteinencoded by an altered immunoglobulin coding region, which may beobtained by expression in a selected host cell. Such altered antibodiesare engineered antibodies (e.g., chimeric or humanized antibodies) orantibody fragments lacking all or part of an immunoglobulin constantregion, e.g., Fv, Fab, Fab′ or F(ab′)₂ and the like. The terms Fv, Fc,Fd, Fab, Fab′ or F(ab′)₂ are used with their standard meanings. See,e.g., Harlow et al. in “Antibodies A Laboratory Manual”, Cold SpringHarbor Laboratory, (1988).

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs or CDRregions in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs or both all heavy and all light chain CDRs, if appropriate.

CDRs provide the majority of contact residues for the binding of theantibody to the antigen or epitope. CDRs of interest in this inventionare derived from donor antibody variable heavy and light chainsequences, and include analogs of the naturally occurring CDRs, whichanalogs share or retain the same antigen binding specificity and/orantagonist ability as the donor antibody from which they were derived,yet may exhibit increased affinity for the antigen. An exemplary processfor obtaining analogs is affinity maturation by means of phage displaytechnology as reviewed by Hoogenboom (1997) Trends in Biotechnology15:62; Barbas et al. (1996) Trends in Biotechnology 14:230; and Winteret al. (1994) Ann. Rev. Immunol. 12:433 and described by Irving et al.(1996) Immunotechnology 2:127.

“Altered immunoglobulin coding region” refers to a nucleic acid sequenceencoding an altered antibody of the invention. When the altered antibodyis a complementarity determining region-grafted (CDR-grafted) orhumanized antibody, the sequences that encode the CDRs from a non-humanimmunoglobulin are inserted into a first immunoglobulin partnercomprising human variable framework sequences. Optionally, the firstimmunoglobulin partner is operatively linked to a second immunoglobulinpartner.

“First immunoglobulin partner” refers to a nucleic acid sequenceencoding a human framework or human immunoglobulin variable region inwhich the native (or naturally-occurring) CDR-encoding regions arereplaced by the CDR-encoding regions of a donor antibody. The humanvariable region can be an immunoglobulin heavy chain, a light chain (orboth chains), an analog or functional fragments thereof. Such CDRregions, located within the variable region of antibodies(immunoglobulins) can be determined by known methods in the art. Forexample Kabat et al. in “Sequences of Proteins of ImmunologicalInterest”, 4th Ed., U.S. Department of Health and Human Services,National Institutes of Health (1987) disclose rules for locating CDRs.In addition, computer programs are known which are useful foridentifying CDR regions/structures.

“Second immunoglobulin partner” refers to another nucleotide sequenceencoding a protein or peptide to which the first immunoglobulin partneris fused in frame or by means of an optional conventional linkersequence (i.e., operatively linked). Preferably, it is an immunoglobulingene. The second immunoglobulin partner may include a nucleic acidsequence encoding the entire constant region for the same (i.e.,homologous, where the first and second altered antibodies are derivedfrom the same source) or an additional (i.e., heterologous) antibody ofinterest. It may be an immunoglobulin heavy chain or light chain (orboth chains as part of a single polypeptide). The second immunoglobulinpartner is not limited to a particular immunoglobulin class or isotype.In addition, the second immunoglobulin partner may comprise part of animmunoglobulin constant region, such as found in a Fab, or F(ab′)₂(i.e., a discrete part of an appropriate human constant region orframework region). Such second immunoglobulin partner may also comprisea sequence encoding an integral membrane protein exposed on the outersurface of a host cell, e.g., as part of a phage display library, or asequence encoding a protein for analytical or diagnostic detection,e.g., horseradish peroxidase, β-galactosidase, etc.

As used herein, an “engineered antibody” describes a type of alteredantibody, i.e., a full-length synthetic antibody (e.g., a chimeric orhumanized antibody as opposed to an antibody fragment) in which aportion of the light and/or heavy chain variable domains of a selectedacceptor antibody are replaced by analogous parts from one or more donorantibodies which have specificity for the selected epitope. For example,such molecules may include antibodies characterized by a humanized heavychain associated with an unmodified light chain (or chimeric lightchain), or vice versa. Engineered antibodies may also be characterizedby alteration of the nucleic acid sequences encoding the acceptorantibody light and/or heavy variable domain framework regions in orderto retain donor antibody binding specificity. These antibodies cancomprise replacement of one or more CDRs (preferably all) from theacceptor antibody with CDRs from a donor antibody described herein.

The term “donor antibody” refers to a monoclonal or recombinant antibodywhich contributes the nucleic acid sequences of its variable regions,CDRs or other functional fragments or analogs thereof to a firstimmunoglobulin partner, so as to provide the altered immunoglobulincoding region and resulting expressed altered antibody with theantigenic specificity and neutralizing activity characteristic of thedonor antibody. Donor antibodies suitable for use in this invention is amurine monoclonal antibody designated as 2C4.

The term “acceptor antibody” refers to monoclonal or recombinantantibodies heterologous to the donor antibody, which contributes all, ora portion, of the nucleic acid sequences encoding its heavy and/or lightchain framework regions and/or its heavy and/or light chain constantregions or V region subfamily consensus sequences to the firstimmunoglobulin partner. Preferably, a human antibody is the acceptorantibody.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one ormore human immunoglobulins. In addition, framework support residues maybe altered to preserve binding affinity. See, e.g., Queen et al. (1089)Proc. Natl. Acad Sci USA 86:10029; Hodgson et al. (1991) Bio/Technology9:421). Furthermore, as described herein, additional residues may bealtered to preserve the activity of the donor antibody.

By “sharing the antigen binding specificity” is meant, for example, thatalthough mAb 2C4 may be characterized by a certain level of bindingactivity, a polypeptide encoding a CDR derived from mAb 2C4 in anyappropriate structural environment may have a lower or higher activity.It is expected that CDRs of mAb 2C4 in such environments willnevertheless recognize the same epitope(s) as mAb 2C4.

The phrase “having the identifying characteristics of” as used hereinindicates that such antibodies or polypeptides share the same antigenbinding specificity as the antibodies exemplified herein, and bind tothe specific antigen with a substantially similar affinity as theantibodies exemplified herein as measured by methods well known to thoseskilled in this art.

A “functional fragment” is a partial heavy or light chain variablesequence (e.g., minor deletions at the amino or carboxy terminus of theimmunoglobulin variable region) which shares the same antigen bindingspecificity as the antibody from which the fragment was derived.

An “analog” is an amino acid sequence modified by at least one aminoacid, wherein said modification can be chemical or a substitution or arearrangement of a few amino acids (i.e., no more than 10) andcorresponding nucleic acid sequences, which modification permits theamino acid sequence to retain the biological characteristics, e.g.,antigen specificity and high affinity, of the unmodified sequence.Exemplary nucleic acid analogs include silent mutations which can beconstructed, via substitutions, to create certain endonucleaserestriction sites within or surrounding CDR-encoding regions.

Analogs may also arise as allelic variations. An “allelic variation ormodification” is an alteration in the nucleic acid sequence encoding theamino acid or peptide sequences of the invention. Such variations ormodifications may be due to degeneracy in the genetic code or may bedeliberately engineered to provide desired characteristics. Thesevariations or modifications may or may not result in alterations in anyencoded amino acid sequence.

The term “effector agents” refers to non-protein carrier molecules towhich the altered antibodies, and/or natural or synthetic light or heavychains of the donor antibody or other fragments of the donor antibodymay be associated by conventional means. Such non-protein carriers caninclude conventional carriers used in the diagnostic field, e.g.,polystyrene or other plastic beads, polysaccharides, e.g., as used inthe BIAcore (Pharmacia) system, or other non-protein substances usefulin the medical field and safe for administration to humans and animals.Other effector agents may include a macrocycle, for chelating a heavymetal atom or radioisotopes. Such effector agents may also be useful toincrease the half-life of the altered antibodies, e.g., polyethyleneglycol.

As used herein, the term “treating” and derivatives thereof meansprophylactic, palliative or therapeutic therapy.

For use in constructing the antibodies, altered antibodies and fragmentsof this invention, a non-human species such as bovine, ovine, monkey,chicken, rodent (e.g., murine and rat) may be employed to generate adesirable immunoglobulin upon presentment with human SAF-2 or a peptideepitope therefrom. Conventional hybridoma techniques are employed toprovide a hybridoma cell line secreting a non-human mAb to SAF-2. Suchhybridomas are then screened for binding activity as described in theExamples section. Alternatively, fully human mAbs can be generated bytechniques known to those skilled in the art.

An exemplary mAb of the present invention is mAb 2C4, a murine antibodywhich can be used for the development of a chimeric or humanizedmolecule. The 2C4 mAb is characterized by specific binding activity onhuman SAF-2. This mAb is produced by the hybridoma cell line 2C4.

The present invention also includes the use of Fab fragments or F(ab′)₂fragments derived from mAbs directed against SAF-2 as bivalentfragments. These fragments are useful as agents having binding activityto SAF-2. A Fab fragment contains the entire light chain and aminoterminal portion of the heavy chain. An F(ab′)₂ fragment is the fragmentformed by two Fab fragments bound by disulfide bonds. The mAb 2C4 andother similar high affinity antibodies provide sources of Fab fragmentsand F(ab′)₂ fragments which can be obtained by conventional means, e.g.,cleavage of the mAb with the appropriate proteolytic enzymes, papainand/or pepsin, or by recombinant methods. These Fab and F(ab′)₂fragments are useful themselves as therapeutic, prophylactic ordiagnostic agents, and as donors of sequences including the variableregions and CDR sequences useful in the formation of recombinant orhumanized antibodies as described herein.

The Fab and F(ab)₂ fragments can be constructed via a combinatorialphage library (see, e.g., Winter et al. (1994) Ann. Rev. Immunol.12:433) or via immunoglobulin chain shuffling (see, e.g., Marks et al.(1992) Bio/Technology 10:779), wherein the Fd or V_(H) immunoglobulinfrom a selected antibody (e.g., 2C4) is allowed to associate with arepertoire of light chain immunoglobulins, V_(L) (or V_(K)), to formnovel Fabs. Conversely, the light chain immunoglobulin from a selectedantibody may be allowed to associate with a repertoire of heavy chainimmunoglobulins, V_(H) (or Fd), to form novel Fabs. Anti-SAF-2 mAbs canbe obtained by allowing the Fd of mAb 2C4 to associate with a repertoireof light chain immunoglobulins. Hence, one is able to recover Fabs withunique sequences (nucleotide and amino acid) from the chain shufflingtechnique.

The mAb 2C4 may contribute sequences, such as variable heavy and/orlight chain peptide sequences, framework sequences, CDR sequences,functional fragments, and analogs thereof, and the nucleic acidsequences encoding them, useful in designing and obtaining variousaltered antibodies which are characterized by the antigen bindingspecificity of the donor antibody.

The nucleic acid sequences of this invention, or fragments thereof,encoding the variable light chain and heavy chain peptide sequences arealso useful for mutagenic introduction of specific changes within thenucleic acid sequences encoding the CDRs or framework regions, and forincorporation of the resulting modified or fusion nucleic acid sequenceinto a plasmid for expression. For example, silent substitutions in thenucleotide sequence of the framework and CDR-encoding regions can beused to create restriction enzyme sites which facilitate insertion ofmutagenized CDR and/or framework regions. These CDR-encoding regions canbe used in the construction of the humanized antibodies of theinvention.

The nucleic and amino acid sequences of the heavy chain variable regionof mAb 2C4 is set forth in SEQ ID NO:1. The CDR amino acid sequencesfrom this region are set forth in SEQ ID NOs: 5, 6 and 7.

The nucleic and amino acid sequences of the light chain variable regionof mAb 2C4 set forth in SEQ ID NO:3. The CDR amino acid sequences fromthis region are set forth in SEQ ID NOs: 8, 9 and 10.

Taking into account the degeneracy of the genetic code, various codingsequences may be constructed which encode the variable heavy and lightchain amino acid sequences and CDR sequences of the invention as well asfunctional fragments and analogs thereof which share the antigenspecificity of the donor antibody. The isolated nucleic acid sequencesof this invention, or fragments thereof, encoding the variable chainpeptide sequences or CDRs can be used to produce altered antibodies,e.g., chimeric or humanized antibodies or other engineered antibodies ofthis invention when operatively combined with a second immunoglobulinpartner.

It should be noted that in addition to isolated nucleic acid sequencesencoding portions of the altered antibody and antibodies describedherein, other such nucleic acid sequences are encompassed by the presentinvention, such as those complementary to the native CDR-encodingsequences or complementary to the modified human framework regionssurrounding the CDR-encoding regions. Useful DNA sequences include thosesequences which hybridize under stringent hybridization conditions tothe DNA sequences. See, T. Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory (1982), pp. 387-389. Anexample of one such stringent hybridization condition is hybridizationat 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for onehour. Alternatively, an exemplary stringent hybridization condition is50% formamide, 4×SSC at 42° C. Preferably, these hybridizing DNAsequences are at least about 18 nucleotides in length, i.e., about thesize of a CDR.

Altered immunoglobulin molecules can encode altered antibodies whichinclude engineered antibodies such as chimeric antibodies and humanizedantibodies. A desired altered immunoglobulin coding region containsCDR-encoding regions that encode peptides having the antigen specificityof an anti-SAF-2 antibody, preferably a high-affinity antibody such asprovided by the present invention, inserted into a first immunoglobulinpartner such as a human framework or human immunoglobulin variableregion.

Preferably, the first immunoglobulin partner is operatively linked to asecond immunoglobulin partner. The second immunoglobulin partner isdefined above, and may include a sequence encoding a second antibodyregion of interest, for example an Fc region. Second immunoglobulinpartners may also include sequences encoding another immunoglobulin towhich the light or heavy chain constant region is fused in frame or bymeans of a linker sequence. Engineered antibodies directed againstfunctional fragments or analogs of human SAF-2 may be designed to elicitenhanced binding with the same antibody.

The second immunoglobulin partner may also be associated with effectoragents as defined above, including non-protein carrier molecules, towhich the second immunoglobulin partner may be operatively linked byconventional means.

Fusion or linkage between the second immunoglobulin partners, e.g.,antibody sequences, and the effector agent, may be by any suitablemeans, e.g., by conventional covalent or ionic bonds, protein fusions,or hetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehydeand the like. Such techniques are known in the art and are described inconventional chemistry and biochemistry texts.

Additionally, conventional linker sequences which simply provide for adesired amount of space between the second immunoglobulin partner andthe effector agent may also be constructed into the alteredimmunoglobulin coding region. The design of such linkers is well knownto those of skill in the art.

In addition, signal sequences for the molecules of the invention may bemodified by techniques known to those skilled in the art to enhanceexpression and intra- and intercellular trafficing.

A preferred altered antibody contains a variable heavy and/or lightchain peptide or protein sequence having the antigen specificity of mAb2C4, e.g., the V_(H) and V_(L) chains. Still another desirable alteredantibody of this invention is characterized by the amino acid sequencecontaining at least one, and preferably all of the CDRs of the variableregion of the heavy and/or light chains of the murine antibody molecule2C4 with the remaining sequences being derived from a human source, or afunctional fragment or analog thereof.

In a further embodiment, the altered antibody of the invention may haveattached to it an additional agent. For example, recombinant DNAtechnology may be used to produce an altered antibody of the inventionin which the Fc fragment or CH2 CH3 domain of a complete antibodymolecule has been replaced by an enzyme or other detectable molecule,i.e., a polypeptide effector or reporter molecule. Other additionalagents include toxins, antiproliferative drugs and radionuclides.

The second immunoglobulin partner may also be operatively linked to anon-immunoglobulin peptide, protein or fragment thereof heterologous tothe CDR-containing sequence having antigen specificity to human SAF-2.The resulting protein may exhibit both antigen specificity andcharacteristics of the non-immunoglobulin upon expression. That fusionpartner characteristic may be, for example, a functional characteristicsuch as another binding or receptor domain or a therapeuticcharacteristic if the fusion partner is itself a therapeutic protein oradditional antigenic characteristics.

Another desirable protein of this invention may comprise a completeantibody molecule, having full length heavy and light chains or anydiscrete fragment thereof, such as the Fab or F(ab′)₂ fragments, a heavychain dimer or any minimal recombinant fragments thereof such as an Fvor a single-chain antibody (SCA) or any other molecule with the samespecificity as the selected donor monoclonal antibody, e.g., mAb 2C4.Such protein may be used in the form of an altered antibody or may beused in its unfused form.

Whenever the second immunoglobulin partner is derived from an antibodydifferent from the donor antibody, e.g., any isotype or class ofimmunoglobulin framework or constant regions, an engineered antibodyresults. Engineered antibodies can comprise immunoglobulin constantregions and variable framework regions from one source, e.g., theacceptor antibody, and one or more (preferably all) CDRs from the donorantibody, e.g., mAb 2C4. In addition, alterations, e.g., deletions,substitutions, or additions, of the acceptor mAb light and/or heavyvariable domain framework region at the nucleic acid or amino acidlevels, or the donor CDR regions may be made in order to retain donorantibody antigen binding specificity.

Such engineered antibodies are designed to employ one (or both) of thevariable heavy and/or light chains of an anti-SAF-2 mAb (optionallymodified as described) or one or more of the heavy or light chain CDRs.The engineered antibodies of the invention exhibit binding activity.

Such engineered antibodies may include a humanized antibody containingthe framework regions of a selected human immunoglobulin or subtype or achimeric antibody containing the human heavy and light chain constantregions fused to the anti-SAF-2 mAb functional fragments. A suitablehuman (or other animal) acceptor antibody may be one selected from aconventional database, e.g., the KABAT® database, Los Alamos database,and Swiss Protein database, by homology to the nucleotide and amino acidsequences of the donor antibody. A human antibody characterized by ahomology to the V region frameworks of the donor antibody or V regionsubfamily consensus sequences (on an amino acid basis) may be suitableto provide a heavy chain variable framework region for insertion of thedonor CDRs. A suitable acceptor antibody capable of donating light chainvariable framework regions may be selected in a similar manner. Itshould be noted that the acceptor antibody heavy and light chains arenot required to originate from the same acceptor antibody.

Preferably, the heterologous framework and constant regions are selectedfrom human immunoglobulin classes and isotypes, such as IgG (subtypes 1through 4), IgM, IgA, and IgE. IgG1, k and IgG4, k are preferred.Particularly preferred is IgG 4, k. Most particularly preferred is theIgG4 subtype variant containing the mutations S228P and L235E (PEmutation) in the heavy chain constant region which results in reducedeffector function. This IgG4 subtype variant is known herein as IgG4PE.See U.S. Pat. Nos. 5,624,821 and 5,648,260.

The acceptor antibody need not comprise only human immunoglobulinprotein sequences. For instance, a gene may be constructed in which aDNA sequence encoding part of a human immunoglobulin chain is fused to aDNA sequence encoding a non-immunoglobulin amino acid sequence such as apolypeptide effector or reporter molecule.

A particularly preferred humanized antibody contains CDRs of mAb 2C4inserted into the framework regions of a selected human antibodysequence. For humanized antibodies, one, two or preferably three CDRsfrom mAb 2C4 heavy chain and/or light chain variable regions areinserted into the framework regions of the selected human antibodysequence, replacing the native CDRs of the human antibody.

Preferably, in a humanized antibody, the variable domains in both humanheavy and light chains have been engineered by one or more CDRreplacements. It is possible to use all six CDRs, or variouscombinations of less than the six CDRs. Preferably all six CDRs arereplaced. It is possible to replace the CDRs only in the human heavychain, using as light chain the unmodified light chain from the humanacceptor antibody. Still alternatively, a compatible light chain may beselected from another human antibody by recourse to conventionalantibody databases. The remainder of the engineered antibody may bederived from any suitable acceptor human immunoglobulin.

The engineered humanized antibody thus preferably has the structure of anatural human antibody or a fragment thereof, and possesses thecombination of properties required for effective therapeutic use such asthe treatment of allergic rhinitis, allergies, asthma, eczema, ordiseases such as lymphoma, leukemia, or systemic mastocytosis.

It will be understood by those skilled in the art that an engineeredantibody may be further modified by changes in variable domain aminoacids without necessarily affecting the specificity and high affinity ofthe donor antibody (i.e., an analog). It is anticipated that heavy andlight chain amino acids may be substituted by other amino acids eitherin the variable domain frameworks or CDRs or both. These substitutionscould be supplied by the donor antibody or consensus sequences from aparticular subgroup.

In addition, the constant region may be altered to enhance or decreaseselective properties of the molecules of this invention. For example,dimerization, binding to Fc receptors, or the ability to bind andactivate complement (see, e.g., Angal et al. (1993) Mol. Immunol.30:105; Xu et al. (1994) J. Biol. Chem. 269: 3469; European PatentPublication No. EP 0 307 434 B1).

An altered antibody which is a chimeric antibody differs from thehumanized antibodies described above by providing the entire non-humandonor antibody heavy chain and light chain variable regions, includingframework regions, in association with human immunoglobulin constantregions for both chains. It is anticipated that chimeric antibodieswhich retain additional non-human sequence relative to humanizedantibodies of this invention may be useful for treating allergicrhinitis, allergies, asthma, eczema, or diseases such as lymphoma,leukemia, or systemic mastocytosis.

Preferably, the variable light and/or heavy chain sequences and the CDRsof mAb 2C4 or other suitable donor mAbs and their encoding nucleic acidsequences, are utilized in the construction of altered antibodies,preferably humanized antibodies, of this invention, by the followingprocess. The same or similar techniques may also be employed to generateother embodiments of this invention.

A hybridoma producing a selected donor mAb, e.g., the murine antibody2C4, is conventionally cloned and the DNA of its heavy and light chainvariable regions obtained by techniques known to one of skill in theart, e.g., the techniques described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory(1989). The variable heavy and light regions containing at least theCDR-encoding regions and those portions of the acceptor mAb light and/orheavy variable domain framework regions required in order to retaindonor mAb binding specificity, as well as the remainingimmunoglobulin-derived parts of the antibody chain derived from a humanimmunoglobulin, are obtained using polynucleotide primers and reversetranscriptase. The CDR-encoding regions are identified using a knowndatabase and by comparison to other antibodies.

A mouse/human chimeric antibody may then be prepared and assayed forbinding ability. Such a chimeric antibody contains the entire non-humandonor antibody V_(H) and V_(L) regions, in association with human Igconstant regions for both chains.

Homologous framework regions of a heavy chain variable region from ahuman antibody are identified using computerized databases, e.g.,KABAT®, and a human antibody characterized by homology to the V regionframeworks of the donor antibody or V region subfamily consensussequences (on an amino acid basis) to mAb 2C4 is selected as theacceptor antibody. The sequences of synthetic heavy chain variableregions containing the CDR-encoding regions within the human antibodyframeworks are designed with optional nucleotide replacements in theframework regions to incorporate restriction sites. This designedsequence is then synthesized using long synthetic oligomers.Alternatively, the designed sequence can be synthesized by overlappingoligonucleotides, amplified by polymerase chain reaction (PCR), andcorrected for errors. A suitable light chain variable framework regioncan be designed in a similar manner.

A humanized antibody may be derived from the chimeric antibody, orpreferably, made synthetically by inserting the donor mAb CDR-encodingregions from the heavy and light chains appropriately within theselected heavy and light chain framework. Alternatively, a humanizedantibody of the invention may be prepared using standard mutagenesistechniques. Thus, the resulting humanized antibody contains humanframework regions and donor mAb CDR-encoding regions. There may besubsequent manipulation of framework residues. The resulting humanizedantibody can be expressed in recombinant host cells, e.g., COS, CHO ormyeloma cells.

A conventional expression vector or recombinant plasmid is produced byplacing these coding sequences for the altered antibody in operativeassociation with conventional regulatory control sequences capable ofcontrolling the replication and expression in, and/or secretion from, ahost cell. Regulatory sequences include promoter sequences, e.g., CMV orRous Sarcoma virus promoter, and signal sequences, which can be derivedfrom other known antibodies. Similarly, a second expression vector canbe produced having a DNA sequence which encodes a complementary antibodylight or heavy chain. Preferably, this second expression vector isidentical to the first except with respect to the coding sequences andselectable markers, in order to ensure, as much as possible, that eachpolypeptide chain is functionally expressed. Alternatively, the heavyand light chain coding sequences for the altered antibody may reside ona single vector.

A selected host cell is co-transfected by conventional techniques withboth the first and second vectors (or simply transfected by a singlevector) to create the transfected host cell of the invention comprisingboth the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The humanized antibody whichincludes the association of both the recombinant heavy chain and/orlight chain is screened from culture by an appropriate assay such asELISA or RIA. Similar conventional techniques may be employed toconstruct other altered antibodies and molecules of this invention.

Suitable vectors for the cloning and subcloning steps employed in themethods and construction of the compositions of this invention may beselected by one of skill in the art. For example, the pUC series ofcloning vectors, such as pUC19, which is commercially available fromvendors such as Amersham or Pharmacia, may be used. Additionally, anyvector which is capable of replicating readily, has an abundance ofcloning sites and selectable genes (e.g., antibiotic resistance), and iseasily manipulated may be used for cloning. Thus, the selection of thecloning vector is not a limiting factor in this invention.

Similarly, the vectors employed for expression of the engineeredantibodies according to this invention may be selected by one of skillin the art from any conventional vector. The vectors also containselected regulatory sequences (such as CMV or Rous Sarcoma viruspromoters) which direct the replication and expression of heterologousDNA sequences in selected host cells. These vectors contain theabove-described DNA sequences which code for the engineered antibody oraltered immunoglobulin coding region. In addition, the vectors mayincorporate the selected immunoglobulin sequences modified by theinsertion of desirable restriction sites for ready manipulation.

The expression vectors may also be characterized by genes suitable foramplifying expression of the heterologous DNA sequences, e.g., themammalian dihydrofolate reductase gene (DHFR). Other preferable vectorsequences include a poly A signal sequence, such as from bovine growthhormone (BGH) and the betaglobin promoter sequence (betaglopro). Theexpression vectors useful herein may be synthesized by techniques wellknown to those skilled in this art.

The components of such vectors, e.g., replicons, selection genes,enhancers, promoters, signal sequences and the like, may be obtainedfrom commercial or natural sources or synthesized by known proceduresfor use in directing the expression and/or secretion of the product ofthe recombinant DNA in a selected host. Other appropriate expressionvectors of which numerous types are known in the art for mammalian,bacterial, insect, yeast and fungal expression may also be selected forthis purpose.

The present invention also encompasses a cell line transfected with arecombinant plasmid containing the coding sequences of the engineeredantibodies or altered immunoglobulin molecules thereof. Host cellsuseful for the cloning and other manipulations of these cloning vectorsare also conventional. However, most desirably, cells from variousstrains of E. coli are used for replication of the cloning vectors andother steps in the construction of altered antibodies of this invention.

Suitable host cells or cell lines for the expression of the engineeredantibody or altered antibody of the invention are preferably mammaliancells such as CHO, COS, a fibroblast cell (e.g., 3T3) and myeloid cells,and more preferably a CHO or a myeloid cell. Human cells may be used,thus enabling the molecule to be modified with human glycosylationpatterns. Alternatively, other eukaryotic cell lines may be employed.The selection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. See, e.g., Sambrook et al.,supra.

Bacterial cells may prove useful as host cells suitable for theexpression of the recombinant Fabs of the present invention (see, e.g.,Plückthun, A., Immunol. Rev., 130, 151-188 (1992)). However, due to thetendency of proteins expressed in bacterial cells to be in an unfoldedor improperly folded form or in a non-glycosylated form, any recombinantFab produced in a bacterial cell would have to be screened for retentionof antigen binding ability. If the molecule expressed by the bacterialcell was produced in a properly folded form, that bacterial cell wouldbe a desirable host. For example, various strains of E. coli used forexpression are well-known as host cells in the field of biotechnology.Various strains of B. subtilis, Streptomyces, other bacilli and the likemay also be employed.

Where desired, strains of yeast cells known to those skilled in the artare also available as host cells, as well as insect cells, e.g.Drosophila and Lepidoptera, and viral expression systems. See, e.g.Miller et al., Genetic Engineering, 8, 277-298, Plenum Press (1986) andreferences cited therein.

The general methods by which the vectors of the invention may beconstructed, the transfection methods required to produce the host cellsof the invention, and culture methods necessary to produce the alteredantibody of the invention from such host cell are all conventionaltechniques. Likewise, once produced, the altered antibodies of theinvention may be purified from the cell culture contents according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like. Such techniques are within the skill ofthe art and do not limit this invention.

Yet another method of expression of the humanized antibodies may utilizeexpression in a transgenic animal, such as described in U.S. Pat. No.4,873,316. This relates to an expression system using the animal'scasein promoter which when transgenically incorporated into a mammalpermits the female to produce the desired recombinant protein in itsmilk.

Once expressed by the desired method, the engineered antibody is thenexamined for in vitro activity by use of an appropriate assay.

Following the procedures described for humanized antibodies preparedfrom mAb 2C4, one of skill in the art may also construct humanizedantibodies from other donor antibodies, variable region sequences andCDR peptides described herein. Engineered antibodies can be producedwith variable region frameworks potentially recognized as “self” byrecipients of the engineered antibody. Modifications to the variableregion frameworks can be implemented to effect increases in antigenbinding and antagonist activity without appreciable increasedimmunogenicity for the recipient. Such engineered antibodies mayeffectively treat a human for ischemic diseases such as myocardialinfarction or cerebral stroke or treatment of vascular insufficiencydiseases, such as diabetes. Such antibodies may also be useful in thediagnosis of those conditions.

This invention also relates to a method for treating allergic rhinitis,allergies, asthma, eczema, or diseases such as lymphoma, leukemia, orsystemic mastocytosis in a mammal, particularly a human, which comprisesadministering an effective dose of a therapeutic agent that binds toSAF-2. Preferred is an anti-SAF-2 monoclonal antibody. The mAb caninclude one or more of the antibodies or altered antibodies describedherein or fragments thereof. Thus, the therapeutic agents of the presentinvention, when in preparations and formulations appropriate fortherapeutic use, are highly desirable for persons susceptible to orexperiencing allergic rhinitis and other allergic diseases, asthma,nasal polyposis, urticaria, hypereosinophilic syndromes (includingChurg-Strauss Syndrome and allergic bronchopulmonary Aspergillosis),eczema, or diseases such as lymphoma, leukemia (including eosinophilicand basophilic leukemias) or systemic mastocytosis.

The monoclonal antibodies used in the methods of the invention caninclude one or more of the antibodies or altered antibodies describedherein or fragments thereof. Preferably, the anti-SAF-2 antibody used inthe methods of the invention has the identifying characteristics of mAb2C4.

The altered antibodies, antibodies and fragments thereof of thisinvention may also be used in conjunction with other antibodies,particularly human mAbs reactive with other markers (epitopes)responsible for the condition against which the engineered antibody ofthe invention is directed.

The antibodies of the present invention can be formulated intopharmaceutical compositions and administered in the same manner asdescribed for mature proteins. See, e.g., International PatentApplication, Publication No. WO90/02762. Generally, these compositionscontain a therapeutically effective amount of an antibody of thisinvention and an acceptable pharmaceutical carrier. Suitable carriersare well known to those of skill in the art and include, for example,saline. Alternatively, such compositions may include conventionaldelivery systems into which protein of the invention is incorporated.Optionally, these compositions may contain other active ingredients.

The therapeutic agents of this invention may be administered by anyappropriate internal route, and may be repeated as needed, e.g., asfrequently as one to three times daily for between 1 day to about threeweeks to once per week or once biweekly. Preferably, the antibody isadministered less frequently than is the ligand, when it is usedtherapeutically. The dose and duration of treatment relates to therelative duration of the molecules of the present invention in the humancirculation, and can be adjusted by one of skill in the art dependingupon the condition being treated and the general health of the patient.

As used herein, the term “pharmaceutical” includes veterinaryapplications of the invention. The term “therapeutically effectiveamount” refers to that amount of therapeutic agent, which is useful foralleviating a selected condition. These therapeutic compositions of theinvention may be administered to mimic the effect of the normal receptorligand.

This invention provides for a pharmaceutical composition which comprisesa therapeutic agent of this invention and a pharmaceutically acceptablecarrier, diluent or excipient. Accordingly, the therapeutic agent may beused in the manufacture of a medicament. Pharmaceutical compositions ofthe therapeutic agent may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation may be a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, standard 5% dextrose in water or bufferedsodium or ammonium acetate solution. Such formulation is especiallysuitable for parenteral administration, but may also be used for oraladministration or contained in a metered dose inhaler or nebulizer forinsufflation. It may be desirable to add excipients such aspolyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride or sodium citrate.

Alternately, the therapeutic agent may be encapsulated, tableted orprepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Solid carriers include starch, lactose, calcium sulfatedihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin,acacia, agar or gelatin. Liquid carriers include syrup, peanut oil,olive oil, saline and water. The carrier may also include a sustainedrelease material such as glyceryl monostearate or glyceryl distearate,alone or with a wax. The amount of solid carrier varies but, preferably,will be between about 20 mg to about 1 g per dosage unit. Thepharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulating, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The mode of administration of the therapeutic agent of the invention maybe any suitable route which delivers the agent to the host. The alteredantibodies, antibodies, engineered antibodies, and fragments thereof,and pharmaceutical compositions of the invention are particularly usefulfor parenteral administration, i.e., subcutaneously, intramuscularly,intravenously or intranasally.

Therapeutic agents of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the engineered (e.g.,humanized) antibody of the invention as an active ingredient in apharmaceutically acceptable carrier. In the compositions of theinvention, an aqueous suspension or solution containing the engineeredantibody, preferably buffered at physiological pH, in a form ready forinjection is preferred. The compositions for parenteral administrationwill commonly comprise a solution of the engineered antibody of theinvention or a cocktail thereof dissolved in an pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., 0.4% saline, 0.3% glycine and the like.These solutions are sterile and generally free of particulate matter.These solutions may be sterilized by conventional, well knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, etc. The concentration of the antibody of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on fluid volumes, viscosities,etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of an engineered antibody ofthe invention. Similarly, a pharmaceutical composition of the inventionfor intravenous infusion could be made up to contain about 250 mL ofsterile Ringer's solution, and about 1 mg to about 30 mg and preferably5 mg to about 25 mg of an engineered antibody of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown or will be apparent to those skilled in the art and are describedin more detail in, for example, “Remington's Pharmaceutical Science”,15th ed., Mack Publishing Company, Easton, Pa.

It is preferred that the therapeutic agent of the invention, when in apharmaceutical preparation, be present in unit dose forms. Theappropriate therapeutically effective dose can be determined readily bythose of skill in the art. Such dose may, if necessary, be repeated atappropriate time intervals selected as appropriate by a physician duringthe response period.

The present invention may be embodied in other specific forms, withoutdeparting from the spirit or essential attributes thereof, and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification or following examples, as indicatingthe scope of the invention.

All publications including, but not limited to, patents and patentapplications, cited in this specification or to which this patentapplication claims priority, are herein incorporated by reference as ifeach individual publication were specifically and individually indicatedto be incorporated by reference herein as though fully set forth.

EXAMPLES

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1 Generation of Monoclonal Antibodies

A. Preparation of Recombinant SAF-2

The full length coding region of SAF-2 (see European Patent PublicationNo. EP 0 924 297 A1, incorporated herein by reference) was subclonedinto the mammalian expression vector pCDN (see Aiyar et al. (1994) Mol.Cell Biochem. 131:75-86) using PCR. The sequence of the insert wasconfirmed before being transfected into HEK293 cells using Ca⁺⁺PO₄.Clones were selected in 500 μg/mL G418 and evaluated for expressionusing Northern blot analysis followed by FACS analysis. Theextracellular domain of SAF-2 was subcloned by PCR and inserted in framewith a Factor Xa cleavage site and the Fc portion of human IgG1. Thesequence was confirmed before the vector was electroporated into CHOEA1cells. Stably expressing clones were selected, expanded, evaluated forFc expression and scaled up. The SAF-2/Fc fusion protein was purifiedfrom supernate using Protein A Sepherose and an aliquot was cleaved withFactor Xa to generate the SAF-2 polypeptide used for antibodygeneration.

B. Monoclonal Antibody Generation

Mice were initially immunized with SAF-2 (25 μg) in Freund's completeadjuvant and then received two booster injections (25 μg) 2 and 4 weekslater. On the basis of a good serum antibody titer to SAF-2, one mousereceived a further immunization of 20 μg of SAF-2 i.v. in PBS. Thespleen was harvested four days later and fused with myeloma cellsaccording to the method described in Zola (Zola, H. (1987) Monoclonalantibodies: A manual of techniques. CRC Press, Boca Raton, Fla.).

C. Hybridoma Screening Assay

Positive hybridomas were tested for binding in 96 well microtiter platescoated with SAF-2/Fc at 0.5 μg/mL and detected with europium conjugatedanti-mouse IgG.

Specifically, 96-well plates were coated with SAF-2/Fc (100 μL/well inPBS) by incubation overnight at 4° C. The solution was then aspiratedand non-specific binding sites were blocked with 250 μL/well of 1%bovine serum albumin (BSA) in TBS buffer (50 mM Tris, 150 mM NaCl, 0.02%Kathon, pH 7.4) for 5-60 minutes at RT. Following this and each of thefollowing steps, the plate was washed 4 times in wash buffer (10 mMTris, 150 mM NaCl, 0.05% Tween 20, 0.02% Kathon, pH 7.4). To each well,50 μL hybridoma medium and 50 μl assay buffer (0.5% BSA, 0.05% bovinegamma globulin, 0.01% Tween 40, 20 μM diethylenetriaminepentaacetic acidin TBS buffer) was added and incubated for 60 minutes at RT in ashaker-incubator. To each well was then added 100 μL 0.5 μg/mL Eu3+labeled anti-mouse antibody in assay buffer. Finally, 200 μL/well ofenhancer (Wallac, Tuku, Finland) was added and incubated for 5 minutesat RT, and the time-resolved fluorescence measured. Positives wererescreened by immunoassay and BIAcore and then cloned by the limitingdilution method. Antibodies produced by cloned cell lines were confirmedto be specific for SAF-2 by ELISA, BIAcore and flow cytometry usingtransfected cell lines.D. Purification of mAbs

Monoclonal antibodies were purified by ProsepA (Bio Processing, Consett,UK) chromatography, respectively, using the manufacturer's instructions.Monoclonal antibodies were >95% pure by SDS-PAGE.

Example 2 Characterization of Monoclonal Antibodies

A. Isotyping of Monoclonal Antibodies

Monoclonal antibody 2C4 used in this study was isotyped as IgG1 kappausing commercially available reagents (Pharmingen, San Diego, Calif.).

B. Affinity Measurements of Monoclonal Antibodies

The affinity of monoclonal antibody 2C4 was determined using a BIAcoreoptical biosensor (Pharmacia Biosensor, Uppsala, Sweden) using a flowrate of 30 μL/min. Kinetic data was evaluated using relationshipsdescribed previously (Karlsson, et al. (1991) J. Immunol. Meth.145:229). The mAb (diluted in HBS buffer, 10 mM HEPES, 150 mM NaCl,0.01% Tween-20, pH 7.4) was injected over a rabbit anti-mouse IgG Fcsurface, followed by buffer flow, and the RU was recorded. SAF-2(diluted in FIBS buffer) was then injected for 180 seconds, followed bya buffer flow for 300 seconds, and the RU was recorded. The sensor chipsurface was regenerated by an injection of 0.1M phosphoric acid. Theon-rates (K_(a)) and off-rates (K_(d)) of binding were calculated usingBIAcore (Uppswala, Sweden) software. The data from this analysisindicated that monoclonal antibody 2C4 displayed an on-rate of (K_(a))2.2×10⁵ M⁻¹s⁻¹ and an off-rate of (K_(d)) 4.3×10⁻⁵ s⁻¹, giving acalculated equilibrium constant (KD) of 2.0×10⁻¹⁰ M.

C. Purification and Culture of Cells

Eosinophils were purified from peripheral blood following Percollremoval of PBMC, lysis of RBC and immunomagnetic negative selection ofneutrophils (Hansel, T. T. et al. (1991) J. Immunol. Methods 145:105).The resulting population was >95% eosinophils. In some experiments,purified eosinophils were cultured for up to two days in complete RPMIcontaining 10% FCS and 1 or 10 ng/mL IL-5, or 10 or 50 ng/mL eotaxin(Peprotech, Rocky Hill, N.J.), C3a, or C5a (Advanced ResearchTechnologies) (Matsumoto, K. et al. (1998) Am. J. Respir. Cell Mol.Biol. 18:860). Viability after 2 days or less of culture was >80%.Enrichment of peripheral blood for basophils was performed using adouble-Percoll density gradient separation, increasing the number ofbasophils to 3-10% of the total leukocyte count (Bochner, B. S. et al.(1989) J. Immunol. Methods 125:265) or with further immunomagneticnegative selection to at least 50%. Human cord blood-derived mast cellswere generated as previously reported (Tachimoto, H. et al. (1997) Int.Arch. Allergy Immunol. 113:293; Saito, H. et al. (1996) J. Immunol.157:343). The purified CD34+ cells were cultured in IMDM supplementedwith 10 μg/mL insulin, 5.5 μg/mL transferrin, 6.7 ng/mL selenium, 5×10-5M 2-mercaptoethanol, 5% fetal bovine serum, 100 U/mL penicillin, 100μg/mL streptomycin, 100 ng/mL stem cell factor (generously provided byAmgen, Thousand Oaks, Calif.) and 50 ng/mL IL-6 (Biosource, Camarillo,Calif.) for at least 10 weeks and 1 ng/mL IL-3 (Biosource) for the first7 days. The purity of mast cells was determined by staining withMay-Grünwald and Giemsa reagents, and routinely reached 99-100% by 14-16weeks of culture. For these experiments, cells used were harvested at16-17 weeks of culture. Bone marrow derived eosinophils were cultured asfollows: light density cells of Ficolled human bone marrow were culturedin IMDM/20% FCS with 20 ng/mL rhGM-CSF and 20 ng/mL rhIL-5 (R&D Systems)at 1.5×106 cells/mL at 37° C., 5% CO2. The cell lines HL-60 and EOL3were treated with sodium butyrate to differentiate them to a moreeosinophil-like phenotype (Collins, S. (1987) Blood 70:1233).

D. Expression of SAF-2 on Human Eosinophils, Basophils and Mast Cells

Expression of integrins or SAF-2 was evaluated in anticoagulated wholeblood or in enriched cells using single color indirect immunofluoresenceand flow cytometry as previously described (Matsumoto, K. et al. (1998)Am. J. Respir. Cell Mol. Biol. 18:860; Bochner, B. S. et al. (1989) J.Immunol. Methods 125:265). Dual color detection of basophils was alsoperformed (Bochner, B. S. et al. (1989) J. Immunol. Methods 125:265).Monoclonal antibodies used included the following: control IgG1, CD18(7E4), CD51 (AMF7, all Coulter-Immunotech, Hialeah, Fla.), CD9(Immunotech) and mAb 2C4. Also used was R-phycoerythrin (PE)-conjugatedor FITC-conjugated F(ab′)2 goat-anti-mouse IgG (Biosource) andFITC-conjugated polyclonal goat anti-human IgE (Kierkegaard and Perry,Gaithersburg, Md.). All samples were fixed in 0.1% paraformaldehyde(Sigma) and analyzed using a FACSCalibur™ flow cytometer (BectonDickinson, Mountainview, Calif.). At least 1,000 events were collectedand displayed on a 4-log scale yielding values for mean fluorescenceintensity (MFI).

SAF-2 was localized to eosinophils (FIG. 3) and was absent from otherpurified cell populations including neutrophils, monocytes, B cells andT cells (data not shown). Activation of purified eosinophils withoptimal concentrations of eotaxin, C5a, C3a or IL-5 for 1 hour, 24 or 48hours before analysis did not alter the levels of SAF-2 expression onthe cell surface (data not shown). Two cells lines, HL-60 and EOL3,which have been reported to become more eosinophil-like followingdifferentiation with Na-butyrate for 5 days, were examined for theexpression of SAF-2 (Mayumi, M. (1992) Leukemia & Lymphoma 7:243). Underthese culture conditions, HL-60 and EOL3 failed to express SAF-2 (datanot shown). Interestingly, when eosinophils are generated in vitro frombone marrow with IL-5, no SAF-2 expression was noted. Eosinophils couldbe identified by day 14 by staining with CD9 (3-12% of the cells) andWright stain (data not shown). It thus appears that SAF-2 expression maybe a later marker for eosinophil differentiation.

Low, but consistently detectable levels of SAF-2 were found on basophils(FIG. 3; for mAb 2C4, 21.1±4.0 percent positive; mean±SEM, n=4). Maturehuman cord blood-derived mast cells also strongly expressed SAF-2,although the pattern of expression was somewhat more heterogeneous thanfor blood leukocytes in that the peaks were not perfectly unimodal (FIG.3).

Example 3 Cloning and Sequencing of Heavy and Light Chain AntigenBinding Regions

Full-length V_(H) and V_(K) region sequences were obtained formonoclonal antibody 2C4 using the following cloning strategy. TheN-terminal amino acid sequences of the mAb 2C4 V_(H) and V_(K) weredetermined. In the event that the N-terminal V region residue wasblocked with pyroglutamic acid, enzymatic de-blocking was performed bymeans of pyroglutamate aminopeptidase.

Total hybridoma RNA was purified, reverse transcribed and PCR amplified.For the heavy chains, the RNA/DNA hybrid was PCR amplified using a mouseIgG CH1-specific primer and a degenerate primer based on the N-terminalprotein sequence. Similarly, for the light chains, the RNA/DNA hybridwas PCR amplified using a mouse C kappa primer and a degenerate primerbased on the N-terminal protein sequence. PCR products of theappropriate size, i.e., ˜350 were cloned into a plasmid vector, andsequenced by a modification of the Sanger method (Sanger et al. (1977)PNAS USA 74:5463). In each case, the sequences of multiple V_(H) clonesand the sequences of multiple V_(K) clones were compared to generate aconsensus heavy chain variable region sequence and consensus light chainvariable region sequence, respectively. The nucleotide and deduced aminoacid sequences of the V_(H) and V_(K) regions of monoclonal antibody 2C4are shown in FIGS. 1 and 2, respectively.

Example 4 Expression of Short (Siglec-8) and Long (Siglec-8L) Forms ofSAF-2 on Eosinophils, Basophils and Mast Cells

-   -   To clarify whether human eosinophils contain Siglec-8 or        Siglec-8L, RT-PCR was performed with Siglec-8-specific or        Siglec-8L-specific primers on mRNA from eosinophils purified as        described previously. Total RNA was prepared with Trizol™ (Life        Technologies, Gaithersburg, Md.) according to the manufacturers'        instructions. After DNase treatment of total RNA (DNA-Free™,        Ambion, Austin, Tex.), cDNAs were synthesized by extension of        oligo(dT) primers (Roche Diagnostics, Indianapolis, Ind.) using        the GeneAmp™ RNA PCR kit (Perkin Elmer, Foster City, Calif.).

Siglec-8 Primers: 5′-CTGCAGGAAGAAATCGGCA-3′ (SEQ ID NO: 11)5′-ATGCTCGGTGTGGAGAAGC-3′ (SEQ ID NO: 12) Siglec-8L Primers:5′-CTGCAGGAAGAAATCGGCA-3′ (SEQ ID NO: 13) 5′-TGTGATTCCTCAAACAGGCCT-3′(SEQ ID NO: 14)

-   -   The amplification cycles were 94° C. for 30 seconds, 60° C. for        45 seconds, and 72° C. for 1 minute. After 35 cycles, PCR        products were separated by 3% agarose gel electrophoresis and        stained with ethidium bromide.        -   Bands for both Siglec-8 and Siglec-8L were detected from            eosinophils. Sequence analysis of these PCR products            revealed a 100% match with those in public databases.        -   Using similar RT-PCR methods, human basophils and HMC-cells            also expressed both Siglec-8 and Siglec-8L mRNA.

Example 5 Functional Analysis

Calcium Flux and Chemotaxis

Initial functional assays (Ca++ and chemotaxis) were performed aspreviously described (Macphee, C. H. et al. (1998) J. Immunol.161:6273). To determine the role of SAF-2 in eosinophil biology,anti-SAF-2 mAbs were analyzed for their ability to affect eosinophilfunction. First, the antibodies were tested for their ability to cause aCa++ flux in purified eosinophils either on their own or followingcrosslinking with a second antibody. Compared with eotaxin, which gave arobust Ca++ response, none of the mAbs to SAF-2 caused a Ca++ flux ineosinophils over a 15 min. time course (data not shown). The mAbs werethen tested for the ability to modulate the Ca++ response to eotaxin inpurified eosinophils. The eosinophils were preincubated with anti-SAF-2with or without a crosslinking antibody and then simulated with eotaxin.Again, the mAbs did not influence the Ca++ flux in response to eotaxin.In addition, the mAbs were also evaluated in an eosinophil chemotaxisassay using eotaxin as the chemotactic agent; again the mAbs failed tomodulate eosinophil function.

Viability Assays

Viability assays were performed in the presence of monoclonal antibodiesdescribed herein. Eosinophils from normal, allergic, andhypereosinophilic donors were purified from peripheral blood asdescribed. Eosinophil purity was consistently >98%, with neutrophilsbeing the only contaminating cells. The viability of freshly isolatedeosinophils was >99% as determined by erythrosin-B dye exclusion.

Polyclonal intact and F(ab′)₂ goat anti-mouse IgG (heavy and lightchain) were purchased from Caltag Laboratories (Burlingame, Calif.).Recombinant human IL-5 and GM-CSF were from R&D Systems (Minneapolis,Minn.). Mouse anti-human CD44 mAb (clone J-173, IgG1), anti-Fas/CD95(clone 7C11, IgM) and anti-CD18 mAb (clone 195N, IgG1) were fromBeckman-Coulter (Hialeah, Fla.). Rabbit polyclonal IgG polyhistidineHis-1 Ab (mHis6 Ab) was from Santa-Cruz Biotechnology (Santa-Cruz,Calif.). IgG and IgM isotype control Abs were from Sigma-Aldrich (St.Louis, Mo.).

Eosinophils were harvested at different time points over 2-72 h afterco-culture with the monoclonal antibodies described herein in thepresence or absence of polyclonal goat anti-mouse IgG Ab used forsecondary cross-linking. In some experiments, intact versus F(ab′)₂ goatanti-mouse IgG were compared in order to elucidate any effect of Fc oneosinophil apoptosis. For controls, cells were incubated with mediumalone or CD44 mAb in the presence or absence of secondary crosslinkingAb and with or without IL-5 or GM-CSF (1-30 ng/ml). In primingexperiments, eosinophils were first preincubated with IL-5 or GM-CSF (30ng/ml) for 24 h, then various Ab were added for an additional 24 h ofculture before analysis of apoptosis.

In certain experiments, viability of cultured eosinophils was determinedby erythrosin dye exclusion as assessed by light microscopy (Matsumoto,K., et al. (1995) Blood 86:1437; Walsh, G. M., et al. (1998) J. Immunol.Methods 217:153). For other experiments, morphological analysis usingestablished light microscopic criteria was performed. Briefly,cytocentrifugation preparations were stained with Leukostat (FisherDiagnostics, Pittsburgh, Pa.) to reveal nuclear morphology. Apoptoticcells were detected by the condensed and rounded appearance of theirnuclei under light microscopy. Cells exhibiting apoptotic nuclei wereenumerated in different fields in a blinded manner using a random codedorder. At least 500 total cells were counted per slide. Cells were thenphotographed using a Zeiss Axioscope microscope (Oberkochen, Germany) at400× magnification. In addition to light microscopic techniques, cellcycle analysis was performed using PI staining (50 mg/ml) of fixed,permeabilized (70% EtOH, 4_(i)C, 30 min), and RNase treated (RNase A,0.05 mg/ml, 37_(i)C, 30 min) eosinophils. Stained cells were thenanalyzed by flow cytometry (FACS Calibur, Becton-Dickinson, San Jose,Calif.) as described previously. Finally, annexin-V labeling was used todetect apoptosis in eosinophils (15, 17).

Using specific murine monoclonal antibodies against Siglec-8 (note thatall subsequent uses of the term Siglec-8 will refer to both isoformsunless specified otherwise) and a secondary polyclonal anti-mouseantibody to enhance crosslinking, we determined whether Siglec-8ligation inhibited eosinophil survival in vitro. As shown in FIG. 4,Siglec-8 crosslinking with 2E2 mAb plus a secondary polyclonal Abinduced a significant increase in eosinophil death. Determined at 24 hof culture, for example, the percentage of eosinophil death induced bySiglec-8 crosslinking (68±4%), was significantly higher then mediumalone (23±4%, p<0.05) or CD44 control crosslinking conditions (9±2%,p<0.0001). The effect of Siglec-8 crosslinking was time dependent, withthe levels of eosinophil death increasing to more than 90% by 48-72 h ofculture.

Eosinophil death induced by Siglec-8 crosslinking, as assessed by dyeexclusion, was already approximately 70% after only 24 h of culture. Tofurther explore the effects of Siglec-8 crosslinking on eosinophil deathand to more carefully examine the kinetics, annexin-V staining was usedto distinguish apoptosis from necrosis at various time points. FIG. 5demonstrates a significant increase in annexin-V+ eosinophils as earlyas 4 h of culture with Siglec-8 crosslinking (15±7%) compared to CD44control crosslinking (5±3%) or medium control (3±1%), indicating a rapidapoptotic effect (n=6). FIG. 4 also demonstrates that the Siglec-8effect became even more pronounced by 24-72 h of culture, especiallywhen compared to effects of the survival promoting cytokine, IL-5. Foran additional assessment of apoptosis induced by Siglec-8 crosslinking,light microscopic examination of eosinophils was performed. After 24 hof culture, Siglec-8 crosslinking on eosinophils resulted in changes inmorphology characterized by reduced cell volume, loss of cytoplasmiccontent, and condensation of nuclei typical of apoptosis. This wasrarely seen in cells cultured with IL-5 or control CD44 crosslinking. Asan average from four experiments, the percentage of eosinophilsdisplaying morphological characteristics of apoptosis with Siglec-8crosslinking was 43±15% compared to 10±2% and 10±1% with medium or CD44Ab crosslinking, respectively. In parallel experiments, we also studiedDNA fragmentation in permeabilized eosinophils, using PI staining infixed cells. Siglec-8 crosslinking for 24 h increased DNA fragmentation(48% hypodiploid DNA staining), compared to 18% and 21% with mediumalone, or CD44 crosslinking, respectively. These data provide multiplelines of evidence demonstrating apoptosis induced by Siglec-8crosslinking.

IL-5 and GM-CSF are potent and specific anti-apoptotic cytokines foreosinophils, and their expression is increased at sites of allergicinflammation in the airways. When eosinophils are cultured in theabsence of survival-promoting cytokines, they rapidly undergo apoptosis;in the presence of these cytokines, their survival can be maintained forweeks. Therefore, we examined the effect of IL-5 and GM-CSF on Siglec-8crosslinking-induced eosinophil apoptosis. When IL-5 (1 ng/ml) was addedsimultaneously with Siglec-8 crosslinking antibodies at the beginning ofthe culture, the cytokine could not override the Siglec-8crosslinking-induced cell death. In fact, at 48 h, the level ofeosinophil apoptosis induced by Siglec-8 crosslinking in the presence of1 ng/ml of IL-5 appeared somewhat higher compared to Siglec-8crosslinking alone (FIG. 6 a, n=4). Similar results were obtained using10 ng/ml of IL-5 or GM-CSF (data not shown). To explore this further, weadded higher concentrations of IL-5 or GM-CSF (30 ng/ml) simultaneouslywith Siglec-8 crosslinking Abs to the initial eosinophil cultures. BothIL-5 and GM-CSF significantly enhanced Siglec-8-induced apoptosis. Thepercentage of apoptosis increased from 53±5% to 74±3% and 76±3% withIL-5 or GM-CSF, respectively (n=4). To determine whether these cytokineswere enhancing eosinophil sensitivity to undergo apoptosis induced bySiglec-8 ligation, experiments were performed in which cells werecultured with saturating to subsaturating concentrations of Siglec-8 mAbin the presence or absence of IL-5, GM-CSF (30 ng/ml) or secondary Ab.Addition of IL-5 or GM-CSF markedly enhanced eosinophil apoptosis evenwhen sub-saturating concentrations of Siglec-8 mAb (0.25 mg/ml) wereused (FIG. 6 b). These data suggest that the presence of IL-5 or GM-CSFrendered eosinophils more sensitive to Siglec-8 crosslinking effectswith respect to its apoptotic effect. Therefore, one additional set ofexperiments was performed to determine whether eosinophil priming withthese cytokines, prior to addition of Siglec-8 crosslinking antibodies,enhanced the pro-apoptotic effect. Eosinophils were preincubated withIL-5 or GM-CSF for 24 h, then mAbs were added and cells were culturedfor an additional 24 h, after which apoptosis was analyzed. Remarkably,cytokine priming (4-30 ng/ml) led to a profound pro-apoptotic responsein the presence of Siglec-8 mAb alone (FIG. 7 and data not shown), aresponse not seen in the absence of cytokines. Note that the percentageof apoptosis in the presence of IL-5 alone or CD44 control Ab alone was14±3%, and 15±5%, respectively (FIG. 7, n=2). Levels of apoptosis with2E2 mAb alone (69±4%) in IL-5-primed eosinophils were similar to thoseseen in unprimed eosinophils exposed to both 2E2 and secondary Ab(72±5%). Similar results were obtained using GM-CSF (FIG. 7).

Crosslinking of Siglec-8 on eosinophils isolated after allergenchallenge of the lower airways by bronchoalveolar lavage using theinstant monoclonal antibodies caused apoptosis of those eosinophils,both in the presence and absence of a secondary, crosslinking antibody(FIG. 8).

The functional consequences of Siglec-8 crosslinking in human basophilsand mast cells was also explored. Culture of human basophils undercrosslinking conditions resulted in reductions in total cellularhistamine content as well as IgE-dependent histamine release responses.Although apoptosis was not specifically studied in this preliminaryexperiment, the most likely explanation for the results observed isinduction, by Siglec-8 crosslinking, of apoptosis and subsequentnecrosis during culture, as well as reduced IgE-dependent releasability.In separate experiments, HMC-1 cells subjected to Siglec-8 crosslinkingfor 72 hours displayed enhanced apoptosis as determined by annexin-Vstaining (47% apoptosis compared to 26% apoptosis under controlconditions, means of n=2). These data suggest that crosslinking ofSiglec-8 may have profound consequences on mast cell and basophilfunction and survival.

1. A method for treating a disease or condition mediated by cellsexpressing SAF-2, the method comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an antibody that binds to an extracellular domain of SAF-2,wherein the disease or condition is systemic mastocytosis, eosinophilicleukemia, or basophilic leukemia.
 2. The method of claim 1, wherein theantibody comprises immunoglobulin complementarity determining regionsset forth in SEQ ID NOs: 5, 6, 7, 8, 9 and
 10. 3. The method of claim 1,wherein the antibody comprises a heavy chain variable region polypeptideas set forth in SEQ ID NO:2 and a kappa light chain variable regionpolypeptide as set forth in SEQ ID NO:4.
 4. The method of claim 1,wherein the antibody comprises a heavy chain variable region polypeptideas set forth in SEQ ID NO:2.
 5. The method of claim 1, wherein theantibody comprises a kappa light chain variable region polypeptide asset forth in SEQ ID NO:4.
 6. The method of claim 1, wherein the antibodyis a humanized antibody.
 7. The method of claim 1, wherein the antibodyis a chimeric antibody.
 8. The method of claim 1, wherein the antibodyis an antibody fragment.
 9. The method of claim 8, wherein the antibodyfragment is selected from the group consisting essentially of an Fvfragment, an Fab fragment, an Fab′ fragment, and an F(ab′)₂ fragment.